Recombinant antibody-based vaccines : A study on the role of asparagine residues as a part of or flanking T cell epitopes

Abstract

There is a fine balance between Ag presentation and by the destruction of Ag peptides. In a “Troybody” vaccine design it is of great importance that the introduced epitopes are properly excised from the Ab molecule. Specific proteases can affect vaccine processing, and the contribution of AEP on processing of the recombinant Ab molecules remains to be elucidated. The aa 89-105 sequence of ë2315 has been introduced in all loops in all constant domains of a human IgG3. Secretion was observed for all mutants except one, loop 2 in CH1. All recombinant Ab with aa 89-105 introduced in CH2 and CH3 induced T cell activation. Only one mutant with aa 89-105 introduced in CH1 induced T cell activation. We could not find an obvious reason for the fact that the peptide is presented from all positions in CH2 and CH3, and not from loop 1, 3, and 5 in CH1. Prediction of AEP cleavage sites within every mutated hIgG3 H chain was performed with NetAEP (http://theory.bio.uu.nl/kesmir/AEP), provided by Can Kesmir at Utrecht University, The Netherlands. This program revealed more restriction sites in the CH2 and CH3 domains than in the CH1 domain. Earlier studies had also shown that the OVA peptide neither was able be presented from LOOP 1 in CH1. We therefore decided to focus on AEP in LOOP 1 in CH1. Both the OVA and the ë2315 peptides contain N in its aa sequence. The deficiency of presentation could be a result of the lack of AEP restriction sites, inaccessibility for AEP restriction sites, or the fact that the epitopes are destructed as shown for the Myelin basic protein (MBP) epitope. A known model epitope, HA, does not contain N. We therefore decided to introduce HA in loop 1 in CH1. Additionally, recognition site for AEP was introduced C-terminally for all three epitopes. Both ë2315 epitopes was included in the study. We found the HA epitope to be presented from loop 1 in CH1, but none of the other two, ë2315 or OVA, to be. Presentation of HA was not enhanced by the proximity of the introduced AEP recognition site. This may be explained by AEP cleavage to occur in another position in the CH1 domain, and the ë2315 and OVA epitopes to be unable of presentation because they are destructed. Alternatively, the mechanism of AEP does not affect the observed results. To prepare comparing studies in a negative cell line, the mouse fibroblast cell line CA.36.2.1 was studied for expression of AEP. We found this cell line to express AEP. An alternative cell must therefore be found to in order to perform this study.